Method for the detection of a polymorphism in cpb2

ABSTRACT

This invention relates to polymorphisms in the human CPB2 gene and corresponding novel allelic polypeptides encoded thereby. The invention also relates to methods and materials for analysing allelic variation in the CPB2 gene, and to the use of CPB2 polymorphism in treatment of diseases with CPB2 drugs.

This invention relates to polymorphisms in the human CPB2 gene andcorresponding novel allelic polypeptides encoded thereby. The inventionalso relates to methods and materials for analysing allelic variation inthe CPB2 gene, and to the use of CPB2 polymorphism in treatment ofdiseases with CPB2 transportable drugs.

Human plasma carboxypeptidase B2 (CPB2) is also known ascarboxypeptidase U (CPU) and thrombin activable fibrinolysis inhibitor;the term CPB2 is used herein. It is an unstable basic carboxypeptidasewhich circulates in human plasma in its proenzyme form,procarboxypeptidase B2 (proCPB2). Vanhoof et al (1996) noted thatproCPB2 exhibits affinity for plasminogen, and is converted to itsactive form through the action of thrombin and plasmin. Bazjar et al(1996) noted that the most likely physiological activator of proCPB2 isthe thrombin-thrombomodulin complex. The deduced amino acid sequence ofthe protein reveals a primary translation product very similar totissue-type carboxypeptidases A and B. Eaton et al (1991) cloned thecDNA for proCPB2. The predicted 423 amino acid protein, consists of a22-amino acid signal peptide, a 92-amino acid activation peptide, and a309-amino acid catalytic domain.

When activated by trypsin, it hydrolyzes carboxypeptidase B substrates,hippuryl-Arg and hippuryl-Lys, but not carboxypeptidase A substrates. Itis inhibited by the specific carboxypeptidase B inhibitor(DL-5-guanidinoethyl) mercaptosuccinic acid. Tsai and Drayna (1992)demonstrated that the gene is located on human chromosome 13. The genewas regionalized by Vanhoof et al (1996) using fluorescence in situhybridisation to 13q14.11. No genetic disorders that are likely toinvolve CPB2 have been mapped to the region.

A possible role for CPB2 is in the activation of plasminogen, an enzymewhich catalyzes the deposition and removal of fibrin. A balance betweenthe activities of the coagulation and fibrinolysis cascades is essentialto protect from excessive blood loss upon injury and to maintain bloodfluidity within the vascular system. Imbalances are characterised byeither bleeding or thrombotic tendencies, the latter of which aremanifested as heart attacks and strokes. Inhibition of CPB2 toaccelerate fibrinolysis could be a treatment for thromboembolicdisorders.

One approach is to use knowledge of polymorphisms to help identifypatients most suited to therapy with particular pharmaceutical agents(this is often termed “pharmacogenetics”). Pharmacogenetics can also beused in pharmaceutical research to assist the drug selection process.Polymorphisms are used in mapping the human genome and to elucidate thegenetic component of diseases. The reader is directed to the followingreferences for background details on pharmacogenetics and other uses ofpolymorphism detection: Linder et al. (1997), Clinical Chemistry, 43,254; Marshall (1997), Nature Biotechnology, 15, 1249; InternationalPatent Application WO 97/40462, Spectra Biomedical; and Schafer et al.(1998), Nature Biotechnology, 16, 33.

Clinical trials have shown that patient response to treatment withpharmaceuticals is often heterogeneous. Thus there is a need forimproved approaches to pharmaceutical agent design and therapy.

Point mutations in polypeptides will be referred to as follows: naturalamino acid (using 1 or 3 letter nomenclature), position, new amino acid.For (a hypothetical) example “D25K” or “Asp25Lys” means that at position25 an aspartic acid (D) has been changed to lysine (K). Multiplemutations in one polypeptide will be shown between square brackets withindividual mutations separated by commas.

One polymorphism in the coding sequence of CPB2 has been described indata from the SNP Consortium, TSC0985620. Zhao et al, Thromb Haemost 80,949-55, 1998 disclosed an A147T protein polymorphism under the name TAPI(equivalent to A169T herein) and a silent base change polymorphism atposition 697. The present invention relates to additional polymorphismsin CPB2 and their haplotypes.

All positions herein of polymorphisms in the CPB2 polynucleotide relateto the position in SEQ ID NO 1 unless stated otherwise or apparent fromthe context.

All positions herein of polymorphisms in the CPB2 polypeptide relate tothe position in SEQ ID NO 2 unless stated otherwise or apparent from thecontext.

According to one aspect of the present invention there is provided amethod for the detection of a polymorphism in CPB2 in a human, whichmethod comprises determining the sequence of the human at at least oneof the following polymorphic positions:

-   -   positions 310, 549, 682 and 772 in CPB2 polynucleotide as        defined by the position in SEQ ID NO: 1; and    -   position 177 in CPB2 polypeptide defined by position in SEQ ID        NO: 2.

D at 177 is conserved between man and mouse and is part of a 26 aminoacid sequence conserved between man and mouse suggesting that this is afunctionally important site. Substitution of G for D substitutes asmall, uncharged amino acid in place of a larger negatively chargedamino acid, which is expected to affect properties of the protein.

According to one aspect of the present invention there is provided amethod for the detection of a polymorphism in CPB2 in a human, whichmethod comprises determining the sequence of the human at at least oneof the following polymorphic positions:

-   -   positions 310, 524, 549, 682, 697 and 772 in CPB2 polynucleotide        as defined by the position in SEQ ID NO: 1; and    -   positions 169 and 177 in CPB2 polypeptide defined by position in        SEQ ID NO: 2.

Preferably the method comprises detection of any one of the followingsix haplotypes: 310 524 682 697 772 1059 Haplotype allele allele alleleallele allele allele 1 C G A C C T 2 C A G C C T 3 C G A C T C 4 T G A TC T 5 C G A C C C 6 C A G C C C

In another embodiment, the method preferably comprises detection of anyone of the following four combinations of allelic variant determinedamino acids based on positions identified in SEQ ID NO: 2: 1thr169/thr347 2 ala169/thr347 3 thr169/ile347 4 ala169/ile347

The term human includes both a human having or suspected of having aCPB2 mediated disease and an asymptomatic human who may be tested forpredisposition or susceptibility to such disease. At each position thehuman may be homozygous for an allele or the human may be aheterozygote.

The term polymorphism includes single nucleotide substitution,nucleotide insertion and nucleotide deletion which in tie case ofinsertion and deletion includes insertion or deletion of one or morenucleotides at a position of a gene and corresponding changes in anypolypeptide encoded thereby.

The method for nucleotide sequence detection is preferably one in whichthe sequence is determined by a method selected from amplificationrefractory mutation system and restriction fragment length polymorphism.

The status of the individual may be determined by reference to allelicvariation at any one, two, three, four, five, six, seven, eight or morepositions.

The test sample of nucleic acid is conveniently a sample of blood,bronchoalveolar lavage fluid, sputum, or other body fluid or tissueobtained from an individual. It will be appreciated that the test samplemay equally be a nucleic acid sequence corresponding to the sequence inthe test sample, that is to say that all or a part of the region in thesample nucleic acid may firstly be amplified using any convenienttechnique e.g. PCR, before analysis of allelic variation.

It will be apparent to the person skilled in the art that there are alarge number of analytical procedures which may be used to detect thepresence or absence of variant nucleotides at one or more polymorphicpositions of the invention. In general, the detection of allelicvariation requires a mutation discrimination technique, optionally anamplification reaction and optionally a signal generation system. Table1 lists a number of mutation detection techniques, some based on thePCR. These may be used in combination with a number of signal generationsystems, a selection of which is listed in Table 2. Furtheramplification techniques are listed in Table 3. Many current methods forthe detection of allelic variation are reviewed by Nollau et. al., Clin.Chem. 43, 1114-1120, 1997; and in standard textbooks, for example“Laboratory Protocols for Mutation Detection”, Ed. by U. Landegren,Oxford University Press, 1996 and “PCR”, 2^(nd) Edition by Newton &Graham, BIOS Scientific Publishers Limited, 1997.

Abbreviations: ALEX ™ Amplification refractory mutation system linearextension APEX Arrayed primer extension ARMS ™ Amplification refractorymutation system b-DNA Branched DNA bp base pair CMC Chemical mismatchcleavage COPS Competitive oligonucleotide priming system CPB2 Humanplasma carboxypeptidase B2 DGGE Denaturing gradient gel electrophoresisFRET Fluorescence resonance energy transfer LCR Ligase chain reactionMASDA Multiple allele specific diagnostic assay NASBA Nucleic acidsequence based amplification OLA Oligonucleotide ligation assay PCRPolymerase chain reaction PTT Protein truncation test RFLP Restrictionfragment length polymorphism SDA Strand displacement amplification SNPSingle nucleotide polymorphism SSCP Single-strand conformationpolymorphism analysis SSR Self sustained replication TGGE Temperaturegradient gel electrophoresis

TABLE 1 Mutation Detection Techniques General: DNA sequencing,Sequencing by hybridisation Scanning: PTT*, SSCP, DGGE, TGGE, Cleavase,Heteroduplex analysis, CMC, Enzymatic mismatch cleavage*Note:not useful for detection of promoter polymorphisms.Hybridisation Based

Solid phase hybridisation: Dot blots, MASDA, Reverse dot blots,Oligonucleotide arrays (DNA Chips)

Solution phase hybridisation: Taqman™—U.S. Pat. No. 5,210,015 & U.S.Pat. No. 5,487,972 (Hoffmann-La Roche), Molecular Beacons—Tyagi et al(1996), Nature Biotechnology, 14, 303; WO 95/13399 (Public Health Inst.,New York)

Extension Based: ARMS™, ALEX™—European Patent No. EP 332435 B1 (ZenecaLimited), COPS—Gibbs et al (1989), Nucleic Acids Research, 17, 2347.

Incorporation Based: Mini-sequencing, APEX

Restriction Enzyme Based: RFLP, Restriction site generating PCR

Ligation Based: OLA

Other: Invader assay TABLE 2 Signal Generation or Detection SystemsFluorescence: FRET, Fluorescence quenching, Fluorescence polarisation -United Kingdom Patent No. 2228998 (Zeneca Limited) Other:Chemiluminescence, Electrochemiluminescence, Raman, Radioactivity,Colorimetric, Hybridisation protection assay, Mass spectrometry

TABLE 3 Further Amplification Methods SSR, NASBA, LCR, SDA, b-DNA

TABLE 4 Protein variation detection methods Immunoassay ImmunohistologyPeptide sequencing

Preferred mutation detection techniques include ARMS™, ALEX™, COPS,Taqman, Molecular Beacons, RFLP, and restriction site based PCR and FRETtechniques. Immunoassay techniques are known in the art e.g. A PracticalGuide to BLISA by D M Kemeny, Pergamon Press 1991; Principles andPractice of Immunoassay, 2^(nd) edition, C P Price & D J Newman, 1997,published by Stockton Press in USA & Canada and by Macmillan Referencein the United Kingdom. Histological techniques are described in Theoryand Practice of Histological Techniques by J D Bancroft & A Stevens,4^(th) Edition, Churchill Livingstone,1996. Protein sequencing isdescribed in Laboratory Techniques in Biochemistry and MolecularBiology, Volume 9, Sequencing of Proteins and Peptides, G Allen, 2^(nd)revised edition, Elsevier, 1989.

Particularly preferred methods include ARMS™ and RFLP based methods.ARMS™ is an especially preferred method.

In a further aspect, there is provided use of a method as defined hereinto assess the pharmacogenetics of a CPB2 drug.

Assays, for example reporter-based assays, may be devised to detectwhether one or more of the above polymorphisms affect transcriptionlevels and/or message stability and/or drug binding. Potential drugs maybe screened to test if polymorphic variation in CPB2 has any affect ondrug performance such as, for example, dosage or efficacy.

Individuals who carry particular allelic variants of the CPB2 gene maytherefore exhibit differences in their ability to regulate proteinbiosynthesis under different physiological conditions and will displayaltered abilities to react to different diseases. In addition,differences arising as a result of allelic variation may have a directeffect on the response of an individual to drug therapy. The methods ofthe invention may be useful both to predict the clinical response tosuch agents and to determine therapeutic dose.

In a further aspect, the methods of the invention, are used to assessthe predisposition and/or susceptibility of an individual to diseasesmediated by CPB2. This may be particularly relevant in the developmentcardiovascular disease and the present invention may be used torecognise individuals who are particularly at risk from developing theseconditions.

In a further aspect, the methods of the invention are used in thedevelopment of new drug therapies which selectively target one or moreallelic variants of CPB2. Identification of a link between a particularallelic variant and predisposition to disease development or response todrug therapy may have a significant impact on the design of new drugs.Drugs may be designed to regulate the biological activity of variantsimplicated in the disease process whilst minimising effects on othervariants.

In a further aspect of the invention the presence or absence of variantnucleotides is detected by reference to the loss or gain of, optionallyengineered, sites recognised by restriction enzymes.

Another aspect of the invention provides a polynucleotide comprising atleast 20 bases of the human CPB2 gene and comprising an allelic variantselected from any one of the following: Position in Variant SEQ ID NO 1allele 310 T 549 G 682 A 772 T

According to another aspect of the present invention there is provided apolynucleotide comprising at least 20 bases of the human CPB2 gene andcomprising an allelic variant selected from any one of the following:Position in Variant SEQ ID NO 1 allele 310 T 524 G 549 G 682 A 697 T 772T

Fragments are at least 17 bases, more preferably at least 20 bases, morepreferably at least 30 bases.

A preferred polynucleotide comprises any one of the following six CPB2haplotypes with reference to positions in SEQ ID NO 1: 310 524 682 697772 1059 Haplotype allele allele allele allele allele allele 1 C G A C CT 2 C A G C C T 3 C G A C T C 4 T G A T C T 5 C G A C C C 6 C A G C C C

The invention further provides a nucleotide primer which can detect apolymorphism of the invention.

According to another aspect of the present invention there is providedan allele specific primer capable of detecting a CPB2 gene polymorphismat one or more of the positions as defined herein.

An allele specific primer is used, generally together with a constantprimer, in an amplification reaction such as a PCR reaction, whichprovides the discrimination between alleles through selectiveamplification of one allele at a particular sequence position e.g. asused for ARMS™ assays. The allele specific primer is preferably 17-50nucleotides, more preferably about 17-35 nucleotides, more preferablyabout 17-30 nucleotides.

An allele specific primer preferably corresponds exactly with the alleleto be detected but derivatives thereof are also contemplated whereinabout 6-8 of the nucleotides at the 3′ terminus correspond with theallele to be detected and wherein up to 10, such as up to 8, 6, 4, 2, or1 of the remaining nucleotides may be varied without significantlyaffecting the properties of the primer.

Primers may be manufactured using any convenient method of synthesis.Examples of such methods may be found in standard textbooks, for example“Protocols for Oligonucleotides and Analogues; Synthesis andProperties,” Methods in Molecular Biology Series; Volume 20; Ed. SudhirAgrawal, Humana ISBN: 0-89603-247-7; 1993; 1^(st) Edition. If requiredthe primer(s) may be labelled to facilitate detection.

According to another aspect of the present invention there is providedan allele-specific oligonucleotide probe capable of detecting a CPB2gene polymorphism, preferably at one or more of the positions definedherein.

The allele-specific oligonucleotide probe is preferably 17-50nucleotides, more preferably about 17-35 nucleotides, more preferablyabout 17-30 nucleotides.

The design of such probes will be apparent to the molecular biologist ofordinary skill. Such probes are of any convenient length such as up to50 bases, up to 40 bases, more conveniently up to 30 bases in length,such as for example 8-25 or 8-15 bases in length. In general such probeswill comprise base sequences entirely complementary to the correspondingwild type or variant locus in the gene. However, if required one or moremismatches may be introduced, provided that the discriminatory power ofthe oligonucleotide probe is not unduly affected. The probes of theinvention may carry one or more labels to facilitate detection.

According to another aspect of the present invention there is providedan allele specific primer or an allele specific oligonucleotide probecapable of detecting a CPB2 gene polymorphism at one of the positionsdefined herein.

According to another aspect of the present invention there is provided akit comprising an allele specific oligonucleotide probe of the inventionand/or an allele-specific primer of the invention.

The kit may comprise appropriate packaging and instructions for use inthe methods of the invention. Such kit may further comprise appropriatebuffer(s) and polymerase(s) such as thermostable polymerases, forexample taq polymerase.

In another aspect of the invention, the polymorphisms of this inventionmay be used as genetic markers in linkage studies. This particularlyapplies to the polymorphisms of relatively high frequency. The CPB2 geneis on chromosome 13q14.11. Low frequency polymorphisms may beparticularly useful for haplotyping as described below. A haplotype is aset of alleles found at linked polymorphic sites (such as within a gene)on a single (paternal or maternal) chromosome. If recombination withinthe gene is random, there may be as many as 2^(n) haplotypes, where 2 isthe number of alleles at each SNP and n is the number of SNPs. Oneapproach to identifying mutations or polymorphisms which are correlatedwith clinical response is to carry out an association study using allthe haplotypes that can be identified in the population of interest. Thefrequency of each haplotype is limited by the frequency of its rarestallele, so that SNPs with low frequency alleles are particularly usefulas markers of low frequency haplotypes. As particular mutations orpolymorphisms associated with certain clinical features, such as adverseor abnormal events, are likely to be of low frequency within thepopulation, low frequency SNPs may be particularly useful in identifyingthese mutations (for examples see: Linkage disequilibrium at thecystathionine beta synthase (CBS) locus and the association betweengenetic variation at the CBS locus and plasma levels of homocysteine.Ann Hum Genet (1998) 62:481-90, De Stefano V, Dekou V, Nicaud V, ChasseJ F, London J, Stansbie D, Humphries S E, and Gudnason V; and Variationat the von willebrand factor (vWF) gene locus is associated with plasmavWP:Ag levels: identification of three novel single nucleotidepolymorphisms in the vWF gene promoter. Blood (1999) 93:4277-83,Keightley A M, Lam Y M, Brady J N, Cameron C L, Ullicrap D).

According to another aspect of the present invention there is provided acomputer readable medium comprising at least one novel sequence of theinvention stored on the medium. The computer readable medium may beused, for example, in homology searching, mapping, haplotyping,genotyping or pharmacogenetic analysis.

Another aspect of the invention provides a method of treating a human inneed of treatment with a CPB2 drug in which the method comprises:

i) detection of a polymorphism in CPB2 in the human, which detectioncomprises determining the sequence of the human at one or more of thefollowing positions:

-   -   positions 310, 549, 682 and 772 in CPB2 polynucleotide as        defined by the position in SEQ ID NO: 1; and    -   position 177 in CPB2 polypeptide defined by position in SEQ ID        NO: 2        and determining the status of the human by reference to        polymorphism in CPB2; and

ii) administering an effective amount of the drug.

According to another aspect of the present invention there is provided amethod of treating a human in need of treatment with a CPB2 drug inwhich the method comprises:

i) detection of a polymorphism in CPB2 in the human, which detectioncomprises determining the sequence of the human at one or more of thefollowing positions:

-   -   positions 310, 524, 549, 682, 697 and 772 in CPB2 polynucleotide        as defined by the position in SEQ ID NO: 1; and    -   positions 169 and 177 in CPB2 polypeptide defined by position in        SEQ ID NO: 2.        and determining the status of the human by reference to        polymorphism in the CPB2 gene; and

ii) administering an effective amount of the drug.

Preferably determination of the status of the human is clinicallyuseful. Examples of clinical usefulness include deciding which drug ordrugs to administer and/or in deciding on the effective amount of theCPB2 drug or drugs. The reader is referred to the following PCT patentapplications from AstraZeneca relating to compounds acting at CPB2: WO00/66152, WO 00/66550 and WO 00/66557. The term “CPB2 drug” means thatdrug affecting CPB2 in humans is an important part of a drug exertingits pharmceutical effect in man.

According to another aspect of the present invention there is provideduse of a CPB2 drug in preparation of a medicament for treating a diseasein a human detected as having a polymorphism at one or more of thepositions defined herein. Preferably the disease is cardiovascular.

According to another aspect of the present invention there is provided apharmaceutical pack comprising CPB2 drug and instructions foradministration of the drug to humans tested for a polymorphism at one ormore of the positions defined herein.

Another aspect of the invention provides an allelic variant of humanCPB2 polypeptide comprising a glycine at position 177 of SEQ ID NO 2; ora fragment thereof comprising at least 10 amino acids provided that thefragment comprises at least one allelic variant.

D at 177 is conserved between man and mouse and is part of a 26 aminoacid sequence conserved between man and mouse suggesting that this is afunctionally important site. Substitution of G for D substitutes asmall, uncharged amino acid in place of a larger negatively chargedamino acid in a conserved region which is expected to affect propertiesof the protein.

According to another aspect of the present invention there is providedan allelic variant of human CPB2 polypeptide comprising at least one ofthe following:

-   -   an alanine at position 169 of SEQ ID NO 2;    -   a glycine at position 177 of SEQ ID NO 2;    -   or a fragment thereof comprising at least 10 amino acids        provided that the fragment comprises at least one allelic        variant.

Fragments of polypeptide are at least 10 amino acids, more preferably atleast 15 amino acids, more preferably at least 20 amino acids.

According to another aspect of the present invention there is providedan antibody specific for an allelic variant of human CPB2 polypeptide asdescribed herein.

Antibodies can be prepared using any suitable method. For example,purified polypeptide may be utilized to prepare specific antibodies. Theterm “antibodies” is meant to include polyclonal antibodies, monoclonalantibodies, and the various types of antibody constructs such as forexample F(ab′)₂, Fab and single chain Fv. Antibodies are defined to bespecifically binding if they bind the allelic variant of CPB2 with aK_(a) of greater than or equal to about 10⁷ M⁻¹. Affinity of binding canbe determined using conventional techniques, for example those describedby Scatchard et al., Ann. N.Y. Acad. Sci., 51:660 (1949).

Polyclonal antibodies can be readily generated from a variety ofsources, for example, horses, cows, goats, sheep, dogs, chickens,rabbits, mice or rats, using procedures that are well-known in the art.In general, antigen is administered to the host animal typically throughparenteral injection. The immunogenicity of antigen may be enhancedthrough the use of an adjuvant, for example, Freund's complete orincomplete adjuvant. Following booster immunizations, small samples ofserum are collected and tested for reactivity to antigen. Examples ofvarious assays useful for such determination include those described in:Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold SpringHarbor Laboratory Press, 1988; as well as procedures such ascountercurrent immuno-electrophoresis (CIEP), radioimmunoassay,radioimmunoprecipitation, enzyme-linked immuno-sorbent assays (ELISA),dot blot assays, and sandwich assays, see U.S. Pat. Nos. 4,376,110 and4,486,530.

Monoclonal antibodies may be readily prepared using well-knownprocedures, see for example, the procedures described in U.S. Pat. Nos.RE 32,011, 4,902,614, 4,543,439 and 4,411,993; Monoclonal Antibodies,Hybridomas: A New Dimension in Biological Analyses, Plenum Press,Kennett, McKearn, and Bechtol (eds.), (1980).

The monoclonal antibodies of the invention can be produced usingalternative techniques, such as those described by Alting-Mees et al.,“Monoclonal Antibody Expression Libraries: A Rapid Alternative toHybridomas”, Strategies in Molecular Biology 3: 1-9 (1990) which isincorporated herein by reference. Similarly, binding partners can beconstructed using recombinant DNA techniques to incorporate the variableregions of a gene that encodes a specific binding antibody. Such atechnique is described in Larrick et al., Biotechnology, 7: 394(1989).

Once isolated and purified, the antibodies may be used to detect thepresence of antigen in a sample using established assay protocols, seefor example “A Practical Guide to ELISA” by D. M. Kemeny, PergamonPress, Oxford, England.

According to another aspect of the invention there is provided a kitcomprising an antibody of the invention.

According to another aspect of the invention there is provided a humanCPB2 polypeptide comprising one of the following four combinations ofallelic variant determined amino acids based on positions identified inSEQ ID NO: 2: 1 thr169/thr347 2 ala169/thr347 3 thr169/ile347 4ala169/ile347

According to another aspect of the invention there is provided apolynucleotide which encodes any of the four combinations of allelicvariant human CPB2 polypeptide as defined above.

References

Eaton, D. L.; Malloy, B. B.; Tsai, S. P.; Henzel, W.; Drayna, D.:Isolation, molecular cloning, and partial characterization of a novelcarboxypeptidase B from human plasma. J. Biol. Chem. 266,21833-21838,1991.

Tsai, S. P.; Drayna, D: The gene encoding human plasma carboxypeptidaseB (CPB2) resides on chromosome 13. Genomics 14, 549-550, 1992.

Vanhoof, G.; Wauters, J.; Schatteman, K.; Hendriks, D.; Goossens, F.;Bossuyt, P.; Scharpe, S.: The gene for human carboxypeptidase U (CPB2)—aproposed novel regulator of plasmingogen activation—maps to 13q14.11.Genomics 38:454-455, 1996.

Bazjar, L.; Morser, J.; Nesheim, E.; TAFI, or plasma procarboxypeptidaseB, couples the coagulation and fibrinolytic cascades through thethrombin—thrombomodulin complex. J. Biol. Chem. 271, 16603-16608, 1996.

The invention will now be illustrated but not limited by reference tothe following Examples. All temperatures are in degrees Celsius.

In the Examples below, unless otherwise stated, the followingmethodology and materials have been applied.

AMPLITAQ™ available from Perkin-Elmer Cetus, is used as the source ofthermostable DNA polymerase.

General molecular biology procedures can be followed from any of themethods described in “Molecular Cloning—A Laboratory Manual” SecondEdition, Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory,1989) or “Current Protocols in Molecular Biology”, Volumes 1-3, Editedby F M Asubel, R Brent & R E Kingston, published by John Wiley, 1998.

Electropherograms were obtained in a standard manner: data was collectedby ABI377 data collection software and the wave form generated by ABIPrism sequencing analysis (2.1.2).

EXAMPLE 1

Identification of Polymorphisms

1.1 Methods

The genomic structure of CPB2 was determined by BLAST analysis using thecDNA sequence against human genomic sequence. This indicated 11 exons.Primers were designed in the flanking intron sequence to amplifyindividual exons from genomic DNA of 29 unrelated Europeans. The PCRprimers were tagged with either M13f or M13r sequence. Individual PCRproducts were sequenced by dye-terminator sequencing in both directionsusing M13f or M13r primers.

1.2 Results Position in Amino acid SEQ ID Reference Variant Variantallele Change, position in NO 1 allele allele Frequency SEQ ID NO 2 310C T 13/56 Silent asp97 524 A G 31/46 Thr169ala 549 A G  1/46 Asp177gly682 G A 35/50 Silent pro 221 697 C T 11/50 Silent asp226 772 C T  4/56Silent ile 251

D at 177 is conserved between man and mouse and is part of a 26 aminoacid sequence conserved between man and mouse suggesting that this is afunctionally important site. Substitution of G for D substitutes asmall, uncharged amino acid in place of a larger negatively chargedamino acid, which is expected to affect properties of the protein.

This analysis also confirmed TSC SNP TSC0985620 as a genuinepolymorphism, substituting T for C at position 1059 and resulting inamino acid change thr347ile. The frequency of the T allele in 40Europeans was 78%.

EXAMPLE 2

Haplotypes

The polymorphisms at 310, 524, 682, 697, 772, and 1059 are inherited as6 common haplotypes. 310 524 682 697 772 1059 Haplotype allele alleleallele allele allele allele 1 C G A C C T 2 C A G C C T 3 C G A C T C 4T G A T C T 5 C G A C C C 6 C A G C C C

These haplotypes indicate that there are 4 common forms of the CPB2protein: Thr169/thr347 (50%), ala169/thr347 (21%), thr169/ile347 (19%)and ala169/ile347 (10%).

1. A method for the detection of a polymorphism in CPB2 in a human,which method comprises determining the sequence of the human at at leastone of the following polymorphic positions: positions 310, 549, 682 and772 in CPB2 polynucleotide as defined by the position in SEQ ID NO: 1;and position 177 in CPB2 polypeptide defined by position in SEQ ID NO:2.
 2. A method according to claim 1 which comprises detection of any oneof the following six haplotypes: 310 524 682 697 772 1059 Haplotypeallele allele allele allele allele allele 1 C G A C C T 2 C A G C C T 3C G A C T C 4 T G A T C T 5 C G A C C C 6 C A G C C C


3. Use of a method as defined in claim 1 to assess the pharmacogeneticsof a CPB2 drug.
 4. A polynucleotide comprising at least 20 bases of thehuman CPB2 gene and comprising an allelic variant selected from any oneof the following: Position in Variant SEQ ID NO 1 allele 310 T 549 G 682A 772 T


5. A polynucleotide according to claim 4 comprising any one of thefollowing CPB2 haplotypes with reference to positions in SEQ ID NO 1:310 524 682 697 772 1059 Haplotype allele allele allele allele alleleallele 1 C G A C C T 2 C A G C C T 3 C G A C T C 4 T G A T C T 5 C G A CC C 6 C A G C C C


6. A nucleotide primer which can detect a polymorphism as defined inclaim
 1. 7. An allele specific primer capable of detecting a CPB2 genepolymorphism defined in claim
 1. 8. An allele-specific oligonucleotideproA polynucleotide comprising at least 20 bases of the human CPB2 geneand comprising an allelic variant selected from any one of thefollowing: Position in Variant SEQ ID NO 1 allele 310 T 549 G 682 A 772T


9. Use of a CPB2 polymorphism as defined in claim 1 as a genetic markerin a linkage study.
 10. A method of treating a human in need oftreatment with a CPB2 drug in which the method comprises: i) detectionof a polymorphism in CPB2 in the human, which detection comprisesdetermining the sequence of the human at one or more of the followingpositions: positions 310, 549, 682 and 772 in CPB2 polynucleotide asdefined by the position in SEQ ID NO: 1; and position 177 in CPB2polypeptide defined by position in SEQ ID NO: 2  and determining thestatus of the human by reference to polymorphism in CPB2; and ii)administering an effective amount of the drug.
 11. An allelic variant ofhuman CPB2 polypeptide comprising a glycine at position 177 of SEQ ID NO2; or a fragment thereof comprising at least 10 amino acids providedthat the fragment comprises at least one allelic variant.
 12. Anantibody specific for an allelic variant of human CPB2 polypeptide asdefined in claim 11.